A Binding Domain on Mesothelin for CA125/MUC16

Ovarian cancer and malignant mesothelioma frequently express both mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The interaction between mesothelin and CA125 may facilitate the implantation and peritoneal spread of tumors by cell adhesion, whereas the detailed nature of this interaction is still unknown. Here, we used truncated mutagenesis and alanine replacement techniques to identify a binding site on mesothelin for CA125. We examined the molecular interaction by Western blot overlay assays and further quantitatively analyzed by enzyme-linked immunosorbent assay. We also evaluated the binding on cancer cells by flow cytometry. We identified the region (296–359) consisting of 64 amino acids at the N-terminal of cell surface mesothelin as the minimum fragment for complete binding activity to CA125. We found that substitution of tyrosine 318 with an alanine abolished CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with alanine could partially decrease binding to CA125, whereas mutation of histidine 354 had no effect. These results indicate that a conformation-sensitive structure of the region (296–359) is required and sufficient for the binding of mesothelin to CA125. In addition, we have shown that a single chain monoclonal antibody (SS1) recognizes this CA125-binding domain and blocks the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain significantly inhibits cancer cell adhesion and merits evaluation as a new therapeutic agent for preventing or treating peritoneal malignant tumors.Ovarian cancer largely is confined to the peritoneal cavity for much of its natural history (1). Peritoneal mesothelioma is a highly invasive tumor originating from the mesothelial linings of the peritoneum (2). The development of effective drug regimens against ovarian cancer and mesothelioma has proven extremely difficult.Mesothelin was first identified in 1992 by the monoclonal antibody (mAb)² K1 that was generated by the immunization of mice with human ovarian carcinoma (OVCAR-3) cells (3). The mesothelin gene encodes a 71-kDa precursor protein that is processed to a 40-kDa protein termed mesothelin, which is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein present on the cell surface (4). Mesothelin is a differentiation antigen that is present on a restricted set of normal adult tissues such as the mesothelium. In contrast, it is overexpressed in a variety of cancers including mesothelioma, ovarian cancer, and pancreatic cancer (5). In addition, mesothelin is also expressed on the surface of non-small cell lung cancer cells (6, 7), especially most lung adenocarcinomas (8).We and others have shown that mesothelin is shed from tumor cells (9, 10), and antibodies specific for mesothelin are elevated in the sera of patients with mesothelioma and ovarian cancer (11). Shed serum mesothelin has been approved by the United States Food and Drug Administration (FDA) as a new diagnostic biomarker in mesothelioma. In a Phase I clinical study of an intrapleural interferon-β gene transfer using an adenoviral vector in patients with mesotheliomas, we found that antitumor immune responses targeting mesothelin were elicited in several patients (12). A recent study indicated that anti-mesothelin antibodies and circulating mesothelin relate to the clinical state in ovarian cancer patients (13). Pastan and colleagues (14) developed an immunotoxin (SS1P) with a Fv for mesothelin. Two Phase I clinical trials were completed at the National Cancer Institute (National Institutes of Health, Bethesda, MD) and there was sufficient antitumor activity of SS1P to justify a Phase II trial. A chimeric antibody containing the mouse SS1 Fv for mesothelin was also developed and is currently examined in a Phase I clinical trial for ovarian cancer, mesothelioma, pancreatic cancer, and non-small cell lung cancer (15).Mucins are heavily glycosylated proteins found in the mucus layer or at the cell surface of many epitheliums (16). There are two structurally distinct families of mucins, secreted and membrane-bound forms. CA125 (also known as MUC16) was first identified in 1981 by OC125, a mAb that had been developed from mice immunized with human ovarian cancer cells (17). The first cDNA clones were reported in 2001 (18, 19). CA125 is a very large membrane-bound cell surface mucin, with an average molecular mass between 2.5 and 5 million daltons. It is also heavily glycosylated with both O-linked and N-linked oligosaccharides (20). The peptide backbone of CA125 is composed of the N-terminal region, extensive Ser/Thr/Pro-rich tandem repeats (TR) with 156 amino acids each with both N- and O-glycosylations, a SEA domain with high levels of O-glycosylation and a C-terminal region with a short cytoplasmic tail (19). The SEA domain was first identified as a module commonly found in sea urchin sperm protein, enterokinase and agrin (21, 22). The significance of the SEA domain in CA125 is not clear.CA125 was originally used as a biomarker in ovarian cancer due to its high expression in ovarian carcinomas and that it is shed into the serum (23). A majority (88%) of mesotheliomas are also CA125 positive on the cell membrane (24). It was shown that 25% of peritoneal mesotheliomas have high CA125 expression (25). The intensity of CA125 membranous expression is indistinguishable between ovarian carcinomas and peritoneal mesotheliomas. Gene expression analysis using the SAGE tag data base has shown that mesothelioma has the second highest co-expression of CA125 and mesothelin after ovarian cancer (26). Rump and colleagues (26) have shown that mesothelin binds to CA125 and that this interaction may mediate cell adhesion. Scholler et al. (27) recently showed that CA125/mesothelin-dependent cell attachment could be blocked with anti-CA125 antibodies. Because mesothelin is present on peritoneal mesothelium, there may be an important role for the mesothelin-CA125 interaction in the tumorigenesis of ovarian cancer and mesothelioma in the peritoneal cavity. The mesothelin binding site on CA125 may lie within the 156-amino acid TR units, indicating multimeric binding of mesothelin to CA125. It has been found that the extraordinarily abundant N-glycans on CA125, presumably in the TR region, are required for binding to both glycosylated and non-glycosylated mesothelin (28).Here, we identified the binding site of CA125 on mesothelin by use of truncated mutagenesis and alanine replacement approaches. We measured binding qualitatively by Western blot overlay assays and quantitatively by enzyme-linked immunosorbent assay (ELISA). We also evaluated the interaction of CA125 and mesothelin on cancer cells by flow cytometry. Furthermore, we have shown that a single chain mAb (SS1) recognized the CA125-binding domain and blocked the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain-Fc fusion protein also significantly inhibited cancer cell adhesion. Our results suggest that conformation-sensitive structures of the region (296–359) are required and sufficient for specific binding of mesothelin to CA125. The domain proteins or the antibodies that block the mesothelin-CA125 interaction merit evaluation as new therapeutic agents in treating peritoneal malignant tumors.     

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.     

Chemical Composition and Pharmacological Evaluation and of Toddalia asiatica (Rutaceae) Extracts and Essential Oil by in Vitro and in Silico Approaches.

Toddalia asiatica (L.) Lam. is extensively used in traditional medicinal systems by various cultures. Despite its frequent use in traditional medicine, there is still a paucity of scientific information on T. asiatica growing on the tropical island of Mauritius. Therefore, the present study was designed to appraise the pharmacological and phytochemical profile of extracts (methanol, ethyl acetate and water) and essential oil obtained from aerial parts of T. asiatica. Biological investigation involved the evaluation of in vitro antioxidant and enzyme inhibitory potentials. The chemical profile of the EO was determined using gas chromatography coupled to mass spectrometry (GC/MS) analysis, while for the extracts, the total phenolic (TPC) and flavonoid content were quantified as well as their individual phenolic compounds by LC/MS/MS. Quinic acid, fumaric acid, chlorogenic acid, quercitrin and isoquercitrin were the main compounds in the extracts. Highest total phenolic (82.5±0.94 mg gallic acid equivalent (GAE/g)) and flavonoid (43.8±0.31 mg rutin equivalent (RE/g)) content were observed for the methanol extract. The GC/MS analysis has shown the presence of 26 compounds with linalool (30.9 %), linalyl acetate (20.9 %) and β-phellandrene (7.9 %) being most abundant components in the EO. The extracts and EO showed notable antioxidant properties, with the methanol extract proved to be superior source of antioxidant compounds. Noteworthy anti-acetylcholinesterase (AChE) and anti-butyrylcholinesterase (BChE) effects were recorded for the tested samples, while only the methanol and ethyl acetate extracts were active against tyrosinase. With respect to antidiabetic effects, the extracts and EO were potent inhibitors of α-glucosidase, while modest activity was recorded against α-amylase. Docking results showed that linalyl acetate has the highest affinity to interact with the active site of BChE with docking score of −6.25 kcal/mol. The findings amassed herein act as a stimulus for further investigations of this plant as a potential source of bioactive compounds which can be exploited as phyto-therapeutics.     

Chromatographic comparison of atenolol separation in reaction media on cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase using ultra fast liquid chromatography

Because chiral liquid chromatography (LC) could become a powerful tool to estimate racemic atenolol quantity, excellent enantiomeric separation should be produced during data acquisition for satisfactory observation of atenolol concentrations throughout the racemic resolution processes. Selection of chiral LC column and analytical protocol that fulfill demands of the ultra fast LC analysis is essential. This article describes the characteristics of atenolol chromatographic separation that resulted from different resolution media and analytical protocols with the use of a Chiralcel® OD column. The chromatograms showed quite different characteristics of the separation process. The single enantiomer and racemic atenolol could be recognized by the Chiralcel® OD column in less than 20 min. Symmetrical peaks were obtained; however, several protocols produced peaks with wide bases and slanted baselines. Observations showed that efficient enantioresolution of racemic atenolol was obtained at slow mobile phase flow rate, decreased concentration of amine‐type modifier but increased alcohol content in mobile phase and highest ultraviolet detection wavelength were required. The optimal ultra fast LC protocol enables to reduce and eliminate the peaks of either the atenolol solvent or the buffers and provided the highest peak intensities of both atenolol enantiomers. Chirality 24:356–367, 2012. © 2012 Wiley Periodicals, Inc.     

The effects of simulated rainfall on immature population dynamics of Aedes albopictus and female oviposition

Larvae of Aedes albopictus Skuse typically inhabit natural and artificial containers. Since these larval habitats are replenished by rainfall, Ae. albopictus may experience increased loss of immature stages in areas with high levels of rainfall. In this study, we investigated the effects of rainfall and container water level on population density, and oviposition activity of Ae. albopictus. In field and laboratory experiments, we found that rainfall resulted in the flushing of breeding habitats. Excess rain negatively impacted larval and pupal retention, especially in small habitats. When filled with water to overflowing, container habitats were significantly repellent to ovipositing females. Taken together, these data suggest that rainfall triggers population loss of Ae. albopictus and related species through a direct detrimental effect (flushing out) and an indirect effect (ovipositional repellency).     

Correction to: Stress impact of liposomes loaded with ciprofloxacin on the expression level of MepA and NorB efflux pumps of methicillin‑resistant Staphylococcus aureus

International Microbiology -     

Tectona grandis L.f: A comprehensive review on its patents,chemical constituents,and biological activities

Tectona grandis L.f is a timber plant that is commonly referred to as teak. Its wide use as a medicine in the various indigenous systems makes it a plant of importance. A wide gamut of phytoconstituents like alkaloids, phenolic glycosides, steroids, etc. has been reported. A renewed interest in this plant has resulted in scientific investigations by various researchers towards the isolation and identification of active constituents along with scientific proof of its biological activities. The different parts of the plant have been scientifically evaluated for their antioxidant, antipyretic, analgesic, hypoglycemic, wound healing, cytotoxic, and many more biological activities. Documentation of this scientific knowledge is of importance to have consolidated precise information encompassing the various aspects of this plant, which could provide a base for future studies. This review is a compilation of the salient reports on these investigations concerning phytochemistry, the methods used to identify and quantify the constituents, the evaluation methods of the biological activity, toxicological studies, allergies and the patent/patent applications. This will further help researchers to find an area of the gap for future studies.     

Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties

The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80-104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55-69 and aa 80-104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody-assisted epitope analysis of N-acetylgalactosaminidase-treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate-binding domain of gC-1.     

Essential trace and toxic element distribution in the scalp hair of Pakistani myocardial infarction patients and controls

The pathogenesis of heart disease has been associated with changes in the balance of certain trace elements. The aim of this study was to evaluate the Zn, Fe, Cu, Cr, Ni, Pb, and Cd contents in scalp hair samples of myocardial infarction (MCI) patients hospitalized in the cardiac ward of National Hospital in Hyderabad city (Pakistan). Scalp hair samples were collected from 193 patients (104 male, 89 female) of 3 age groups (46–60, 61–75, and 76–90 yr), for a comparative study, 200 normal, healthy subjects (103 male, 97 female) of the same age groups residing in the same city were selected. All metals in scalp hair samples were assessed by a flame/graphite furnace atomic absorption spectrophotometer, prior to microwave-assisted and conventional wet acid digestion methods. Results were calculated in micrograms per gram. The mean values of Fe and Zn of scalp hair samples of MCI patients were significantly reduced compared to the control subjects of both genders. The mean Fe concentrations in male patients were 19.42, 12.36, and 6.98 vs 30.69, 24.42, and 16.75 for the control patients in the three age groups (46–60, 61–75, and 76–90 yrs, respectively). The mean Zn concentration in male patients were 169.2, 149.4, and 107.7 μg/g vs 206.1, 188.0, and 154.4 μg/g for the control group (p<0.002, 0.004, and 0.001) in all three age groups, respectively. These differences were also observed in the female study groups. The mean values of Pb, Cd, and Ni were significantly high in patients compared to healthy subjects (mean Pb in male patients: 11.85, 12.89, and 14.52 those of female patients were 11.88, 12.73, and 14.21 vs the male controls patients (6.08, 7.56, and 8.56) and female controls (5.99, 7.41, and 8.25) for all three age groups, respectively. The concentration of Ni and Cd in the scalp hair samples of the heart patients of both sexes were significantly higher compared to the control; in the case of Ni the range of significant difference for males was found to be p<0.001–0.009 and for females to be p<0.0.002–0.007 and significantly high concentration of Cd were observed in hair samples of patients than in controls in the range for males (p<0.001–0.009) and in females (p<0.001–0.011). The Zn/Cu and Zn/Cd ratios in the scalp hair (p<0.01) of the diseased groups were significantly lower than that of the healthy groups. Deficiency of essential trace metals and high level of toxic metals might play a role in the development of heart disease in the subjects of this study. Toxic metals might also cause diminished absorption of essential elements.     

Parasitism,longevity and progeny production of six indigenous Kenyan trichogrammatid egg parasitoids (Hymenoptera: Trichogrammatidae) at different temperature and relative humidity regimes

Six native Kenyan species/strains of Trichogramma and Trichogrammatoidea, recovered from Helicoverpa armigera were evaluated at six different temperatures (10, 15, 20, 25, 30 and 35°C) and two relative humidity levels (40–50 and 70–80%) with the aim of selecting strains adapted to warmer temperature regimes. The species/strains were collected from low (<700 m), medium (between 700 and 1200 m) and high altitude (>1200 m) locations and were evaluated for parasitism, adult longevity, progeny production and progeny sex ratio at the different environmental regimes. Eggs of the factitious host, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) were used in the investigations. Temperature and humidity interactions affected parasitism and progeny production. The highest parasitism at the two humidity levels was at 25 and 30°C for all the strains evaluated. Adult longevity was also significantly affected by the interaction of temperature and relative humidity and was longer at the lower than higher relative humidity. Survival followed a type I survivorship curve at lower temperatures and a type III survivorship curve at the higher temperatures. Trichogramma sp. nr. mwanzai from low altitude, Trichogramma sp. nr. mwanzai from medium altitude and Trichogrammatoidea sp. nr. lutea also from medium altitude lived longer than other strains at all the temperatures and relative humidity levels evaluated, including the warmest regimes of 30 and 35°C. These strains appear promising as candidates for augmentative biocontrol of H. armigera in Kenya.